Complex networks of highly crosslinked biopolymers are common materials in living tissues. Familiar examples of these are the networks of collagen fibers that form the matrices of skin, bones, and connective tissues. The crosslinking found in these materials gives them their tensile strength, elasticity, and other valuable characteristics. In nature, the crosslinking, which is so critical to the function of these tissues, is accomplished by enzymatic oxidation of specific amino acid side chains of the protein materials involved, thus creating reactive intermediates which spontaneously form crosslinks. In the case of collagen and elastin, the ε-amino group of specific lysine residues are oxidatively deaminated by lysyl oxidase into reactive aldehyde groups that spontaneously condense with neighboring peptidyl aldehydes or ε-amino groups (Kagan, H. M. et al., J Cell Biochem, 2003. 88(4): p. 660-72).
Another widely occurring mechanism of biomolecule crosslinking is oxidative coupling through phenols. Examples are the formation of lignin, which is a major structural component of plant cell walls, the formation of melanin pigments, the curing of tree sap into hard lacquers, the formation of peptidic natural products that exhibit high biological activity, such as vancomycin (Jung, G., Letters in Peptide Science, 2001. 8(3-5): p. 259-265; Malnar, I. et al. Tetrahedron Letters, 2000. 41(12): p. 1907-1911; Nishiyama, S., et al., Tetrahedron Letters, 1994. 35(45): p. 8397-8400) and bouvardin (Boger, D. L., et al. JACS, 1994. 116(19): p. 8544-56), through the formation of dityrosine protein crosslinks. Dityrosine protein linkages have been found in many structural proteins including elastin (LaBella, F., et al., Biochem Biophys Res Commun., 1967. 26(6): p. 748-53; Malanik, V. et al., Connect Tissue Res, 1979. 6(4): p. 235-40), silk (Raven, D. J., C. Earland, et al., Biochim Diophys Acta, 1971. 251(1): p. 96-99), plant cell wall extensin (Fry, S. C., Biochem J, 1982. 204(2): p. 449-55; Brady, J. D., et al. Biochem J, 1996. 315(Pt 1): p. 323-7), and in hardened fertilization membranes of insect and sea urchin eggs (Foerder, C. A. et al., PNAS (USA), 1977. 74(10): p. 4214-8). These oxidative phenolic coupling processes are catalyzed by a large number of structurally and mechanistically distinct metalloenzymes, including peroxidases, tyrosinases, and laccases. Peroxidases crosslink tyrosines by extracting a single electron from the phenol side chain to create a radical that then reacts with a vicinal phenol side chain to form dityrosine or isodityrosine (Michon, T., et al., Biochemistry, 1997. 36(28): p. 8504-13; Eickhoff, H., et al., Tetrahedron, 2001. 57(2): p. 353-364). Tyrosinases may crosslink tyrosine residues in two steps: first, the phenol side chain is hydroxylated forming 3,4-dihydroxyphenyl-L-alanine (DOPA); second, DOPA is further oxidized to form reactive o-quinones that spontaneously crosslink (Sanchez-Ferrer, A., et al., Biochim Biophys Acta, 1995. 1247(1): p. 1-11; Espin, J. C., et al., Eur J Biochem, 2000. 267(5): p. 1270-9).
These natural oxidative protein crosslinking mechanisms are mediated by transition metal-peptidyl coordination complexes. The redox active metal complexes come in many forms. Lysyl oxidase contains a single copper (II) atom and an organic co-factor formed by the intermolecular crosslinking of a lysine ε-amine and a tyroxyl quinone (Kagan, H. M. et al., J Cell Biochem, 2003. 88(4): p. 660-72). Tyrosinases (Sanchez-Ferrer, A., et al., Biochim Biophys Acta, 1995. 1247(1): p. 1-11) and laccases (Thurston, C. F., Microbiology, 1994. 140: p. 19-26) are multi-copper enzymes that transfer electrons to molecular oxygen. Peroxidases that crosslink tyrosines are iron-heme proteins that transfer single electrons from phenols to H2O2 (Michon, T., et al., Biochemistry, 1997. 36(28): p. 8504-13; Oudgenoeg, C., et al., J. Agric. Food Chem., 2001. 49(5): p. 2503-10). Nature has found many distinct metal-mediated pathways for extracting electrons to crosslink phenolic compounds.
Another particularly intriguing example of crosslinking through tyrosine derivatives occurs in mussel byssal threads, which are specialized ligaments that bond the animals to an external substrate. The byssal threads contain several proteins and are heavily diDOPA crosslinked (Rzepecki, L. M., et al., Mol Mar Bid Biotechmol, 1991. 1(1): p. 78-88; McDowell, L. M., et al., J Biol Chem, 1999. 274(29): p. 20293-5). The thread proteins include three unusual collagens with blocky primary structures; a central collagen block is flanked by either elastin-like blocks (pre-ColP) (Coyne, K. J., et al., Science, 1997. 277(5333) p. 1830-2), silk-like blocks (pre-ColD) (Qin, X X., et al., J Biol Chem, 1997. 272(51): p. 32623-7), or amorphous glycine-rich blocks (pre-ColNG) (Qin, X X. et al., PNAS (USA), 1998. 95(18): p. 10517-22). All three have histidine- and DOPA-rich domains at the amino- and carboxyl-termini. The histidine blocks likely chelate the several metal ions found associated with byssal threads. Chelate complexes are thought to participate in non-covalent intermolecular crosslinking of the collagens (Vaccaro, F. et al., Biomacromolecules, 2001. 2(3): p. 906-11).
Chelated transition metal ions, particularly Ni(II), can participate in oxidative crosslinking of biomolecules. Much of the research in this area has been done from the perspective of understanding nickel toxicity, which may be largely due to its oxidative activity when chelated by peptides, proteins, or nucleic acids (Gill, G., et al., Chem Res Toxicol, 1997. 10(3): p. 302-9; Bal, W., et al., Chem Res Toxicol, 2000. 13(7): p. 616-24). A second area of research has focused on creating synthetic catalysts that mimic metalloenzymes. For example, tyrosinase model complexes have been synthesized that are capable of regiospecific oxidation of phenol and catechol (Monzani, E., et al. Inorganic Chemistry, 1998. 37: p. 553-562) and regiospecific polymerization of phenol into poly(1,4-phenylene oxide) (Higashimura, H., et al. J. Am. Chem. Soc., 1998. 120: p. 8529-8530). A third area of research has been directed at exploiting the oxidative activity of chelated Ni(II) to create reagents for in vitro conjugation, crosslinking, or cleavage of biomolecules. A number of metal complexes have been synthesized that specifically modify DNA (Muller, J. G., et al., J Inorg Biochem, 1994. 54(3): p. 199-206; Burrows, C. J., et al., Acta Chem Scand, 1996. 50(4): p. 337-44; Hickerson, R. P., et al., J Mol Biol, 1998. 279(3): p. 577-87; Stemmler, A. J. et al.: JACS, 1999. 121(29): p. 6956-6957).
With regard to protein modification, others have found that Ni(II) complexed by the tripeptide NH2-Gly-Gly-His (GGH) in the presence of the relatively strong oxidant monoperoxyphthalic acid (MMPP) crosslinked proteins known to associate in solution (Brown, K. C., et al., Biochemistry, 1995. 34(14): p. 4733-9). Subsequently, GGH genetically appended to the dimeric protein, ecotin, mediated intermolecular crosslinking between tyrosine residues in the presence of MMPP and Ni(II) (Brown, K. C., et al., Biochemistry, 1998. 37(13): p. 4397-406). Crosslinking between wild-type ecotin, in which tyrosine residues at the dimer interface are separated by 20 Å, was relatively inefficient (15%). By placing tyrosine residues within 5 Å of one another at the dimer interface, crosslinking efficiency was increased to 60%. This demonstrated a major advantage of protein crosslinking mediated by peptide metal complexes, that a redox active Ni(II) chelator and target tyrosine residues can be genetically positioned within a protein, creating the potential for site specific protein modification through oxidative conjugation or crosslinking. A recent report, however, revealed that this chemistry is much more complex than just dityrosine formation. A number of reactions occur in the strong oxidizing environment, and crosslinking between the terminal GG residues and tyrosine is one of the major reactions (Person, M. D., et al., Protein Sci, 2001. 10(8): p. 1549-62).
The His6 tag (SEQ ID NO:1) commonly used for purification of recombinant proteins by immobilized metal affinity chromatography (IMAC) has also been shown to be redox active in the presence of nickel(II) and peracid oxidants. HIS-tagged glutathione S-transferase (H6GST (SEQ ID NO:2)), a dimer in solution, was covalently crosslinked in the presence of Ni(II) and MMPP (Fancy, D. A., et al., Chern Biol, 1996. 3(7): p. 551-9). Proteins that do not naturally associate with H6GST were not crosslinked, demonstrating that His6-mediated crosslinking does not proceed through a highly diffusible reactant and crosslinking is localized to the vicinity of the His6 tag. Going further, the same research group demonstrated that, in the presence of MMPP and Ni(II), H6GST mediated the formation of dityrosine from free tyrosine, that the mutagenic removal of tyrosine residues from H6GST decreased crosslinking efficiency, and that chemical addition of tyrosine residues using the Bolton-Hunter reagent increased crosslinking efficiency (Fancy, D. A., et al., Biochem Biophys Res Commun, 1998. 247(2): p. 420-26). The primary interest of this research appears to be the architectural analysis of multi-protein complexes.
A large and successful industry has grown up around the development of reagents for the post-translational chemical modification of proteins. Despite the ingenious variety of reagents and methods available, there is a continuing need for new protein technologies that grows in proportion to the increasing importance of proteins in several major industries, including pharmaceutics, clinical diagnostics, chemicals, energy, agriculture, environmental protection, food, and textiles processing. New and better tools for labeling, conjugating, crosslinking, and immobilizing proteins would be valuable in all of these industries. The expanding industrial use of proteins, driven by new protein technologies, may have profound effects on human health, ranging from more cost effective pharmaceuticals to decreased environmental and the consequent human health impact from the production of chemicals and energy.
In particular, methods are desired for crosslinking proteins, polymers, and/or other moieties, including those that are not naturally associated, illustratively in a manner that is predictable. Accordingly, a method of crosslinking proteins (such as enzymes), polymers (including biopolymers such as protein, polysaccharide, poly-nucleic acid, lipid, and synthetic polymers such as polyethylene glycol, (“PEG”), polypropylene glycol, polyesters, and polymeric surfaces (such as pluronics)), and/or other moieties (such as fluorescent compounds, small molecules illustratively having a molecular weight from 50 g/mol to 800 g/mol, and small molecules having a degree of polymerization from 1 to about 10), by contact with an oxidant after associating the two species to be linked in a coordination complex with a metal ion or ions is provided. Such a method is useful in a variety of applications, including the labeling of proteins, PEGylating proteins, coupling proteins and polymers, and affixing proteins to surfaces. In some embodiments, the method comprises a method of crosslinking a first and a second protein comprising the steps of attaching a metal-binding peptide, illustratively a HY-tag, to the first protein using methods known in the art; attaching a metal-binding peptide to the second protein using methods known in the art; forming a coordination complex between the first protein, the second protein, and a metal ion; and crosslinking the first and second proteins by exposing the coordination complex to an oxidant. In illustrative embodiments of the instant invention, the metal ion is selected from the group consisting of nickel, copper, cobalt, gadolinium, iron, osmium, palladium, rhodium, ruthenium, samarium, selenium, silver, strontium, tantalum, thulium, tin, tungsten, vanadium, yttrium, and zinc. In others, the metal ion may be a first row transition metal ion. The methods of this invention may be used for crosslinking any combination of proteins, polymers, and small molecules.
An oxidizing agent is a chemical reagent that is an oxidizer, or which promotes the oxidation of a desired reagent. In some embodiments of the invention, MMPP is used to promote the oxidation of tyrosine residues. Other oxidants have been found suitable as well, including peroxides, other peracids, oxidizing enzymes such as tyrosinase, oxygen, and other weaker oxidants, wherein a “weaker oxidant” is weaker than MMPP. In some embodiments, however, a stronger oxidant may be used. Illustratively, the oxidizing agent may be generated from an electrode surface. In one embodiment, the electrode surface is coated with a synthetic or natural polymer. Na2SO3 may also be used. While not itself an oxidant, Na2SO3 auto-oxidizes in the presence of complexed NiH to persulfate (SO5). In another embodiment, the oxidizing agent may be activated by a metalloenzyme, including but not limited to a peroxidase, a tyrosinase, a laccase, and a catechol oxidase. One suitable metalloenzyme is horseradish peroxidase.
In some embodiments, the ligand used to create the coordination complex is a metal-binding peptide or metal chelating tag, such as an HY-tag. In illustrative embodiments of the instant invention, the HY-tag is chosen from a group of sequences that comprise about four to ten histidine residues and a number of tyrosine residues. The sequences largely differ from each other in the placement of the tyrosines. As used herein, the term HIS-tags refers to HIS-tags generally, while HIS-TYR tags or HY-tags refer to HIS-tags that include tyrosines therein. Either may be referred to as his6-tags when the number of histidine residues is specified. Also, it is understood that while HY-tags are used in illustrative embodiments, other chelating constructs comprised of amino acids and/or other natural or synthetic monomers may be used, illustratively including tyrosine, dihydroxyphenylalanine, or other phenolic groups. It is understood that phenolic groups includes compounds having one or more aromatic benzene rings having one or more hydroxyl groups (—OH). In addition, phenolic derivatives, including oxidized forms of phenolic groups, may be used. Phenolic groups and phenolic derivatives may contain further substituents on the benzene ring in addition to the hydroxyl group(s).
Some of the embodiments of the invention may comprise methods for labeling proteins, as when a fluorescent label (illustratively fluorescein, rhodamine, CY-3, and CY-5), enzyme, or other molecule is attached. Other such molecules could include biotin, digoxigenin, polyethylene glycol, pluronics, and other suitable labels. In one embodiment, the second moiety can be a small molecule. In one aspect, the small molecule can have a molecular weight from 50 g/mol to 800 g/mol. In another aspect, the small molecule can be an oligomer with a degree of polymerization from 1 to 10. In still another aspect, the small molecule can be comprised of epitope labels or fluorophores, including those selected from the group consisting of digoxigenin, biotin, fluorescein, rhodamine, CY-3, CY-5, and derivatives thereof.
In some embodiments, the invention comprises a method of conjugating a protein with a synthetic polymer comprising the steps of attaching a HY-tag to a selected protein; attaching a HY-tag or other metal-chelating group containing or in close proximity to a phenolic functional group to a selected polymer; forming a coordination complex between the protein, the polymer, and a metal ion; and crosslinking the protein and polymer by exposing the coordination complex to an oxidant. In various of these embodiments, the polymer is a synthetic polymer. In some, the polymer is polyethylene glycol. In others, the polymer is chosen from the group of polyacrylates, polymer surfaces such as pluronics, carbohydrates such as dextran, and lipids. In still other embodiments, HY-tags, which may be the same or different, are used to link two polymers.
Other embodiments of the instant invention comprise methods of immobilizing a protein or other moiety on a polymer surface under conditions that preserve protein structure and activity, comprising the steps of modifying a polymeric surface such that the polymeric surface comprises a synthetic chelator; attaching a HY-tag to a protein; forming a coordination complex between the polymeric surface, the protein, and a metal ion; and immobilizing the protein to the polymeric surface by exposing the coordination complex to an oxidant, thus causing the crosslinking of the polymer and the protein. In some embodiments, the polymer surface is further comprised of a synthetic polymer. In others, the polymer is polyethylene glycol. In still others, the polymer is a pluronic.
Similarly, the moiety may already be immobilized on a solid surface and the method may be used to link a second moiety to the surface. Examples of solid surfaces include tissues, polymers, metals, ceramics, composites, biopolymers, bioceramics (such as bones and teeth), and colloidal particles. Colloidal particles illustratively include materials gold, silver, silica, semiconductors, fluorescent semiconductors, polystyrene, polymeric micelles, dendrimers, liposomes, and viruses, and may be of any size, illustratively of from 1 nm to 100 μm. The polymer may be coated onto or otherwise bound to the solid surface. It is also understood that the methods of this invention may be used to link two different surfaces together, wherein each of the two moieties is immobilized on its respective surface. Illustratively, this may be useful when each of the surfaces is a tissue, and the crosslinking may function as a surgical glue.
While targets catalyzed by the mildest possible oxidant, illustratively O2, are desired for use with proteins that are particularly sensitive to oxidation, it is understood that stronger oxidants may be used in some instances, and that the metal complex may be selected accordingly. Also, while tyrosines are used to crosslink through dityrosine formation, other phenolic residues may be used and crosslinking may occur through a substituted bis-phenol adduct.
Various features and embodiments of the instant invention are useful for a variety of common research and medical applications. One of these is use in the formation of hydrogels. The ability to provide predictable crosslinking of the proteins in such gels could allow the formation of new types of gels with unique properties and characteristics. This application could also be used to encapsulate cells, proteins, and DNA for preservation and use. A related application would be use of the invention as a surgical glue. Such a glue could be composed of specifically-chosen proteins selected for ability to prevent or avoid an immune response, promote growth and regeneration of surrounding tissue, degrade over time, or exhibit any other possible characteristic. Additionally, the glue could be applied in a liquid form to the wound, and then the glue could be cured and the wound sanitized simultaneously by the application of hydrogen peroxide as an oxidant/sterilant. Many other uses are possible.
These and other objects, features, and advantages of the present invention will become more fully apparent from the following description and appended claims, or maybe learned by the practice of the invention as set forth hereinafter.